Nearly every cell of the body contains organelles called mitochondria which produce most of the energy used by the body. Certain cells with high metabolic rates, such as heart muscle cells, may contain thousands of mitochondria.
Energy derived from the utilization of substrates is required in order to maintain the non-equilibrium state necessary to carry out the basic functions of the cell (e.g. contraction, secretion, electrical propagation, ion pumping, cell division). There are three major steps involved in cellular energy production. First, large food molecules are broken down into smaller units. Polysaccharides are converted to simple sugars, fat is converted to fatty acids and proteins are converted to amino acids. Second, multiple pathways convert these three different molecules into a common precursor, acetyl CoA, for further oxidation by the mitochondria Third, the Krebs cycle (a.k.a. the citric acid or tricarboxylic acid cycle) and oxidative phosphorylation convert acetyl CoA into ATP, the energy storage molecule directly utilized by the energy consuming reactions in the cell.
Mitochondrial substrate oxidation is a multi-step process that includes a series of reactions which transfer electrons from the initial substrate to nicotinamide adenine dinucleotide (NAD+) to produce NADH which is then reoxidized by passing electrons through the electron transport chain to the ultimate electron acceptor oxygen. In the process of electron transfer, protons (H+) are pumped out of the mitochondrial matrix (an intracellular aqueous compartment bounded by the inner and outer mitochondrial membranes between it and the cell's cytoplasm) across the mitochondrial inner membrane (IMM) resulting in the establishment of a proton gradient. This proton concentration gradient, together with the large membrane voltage generated by the active charge movement across the IMM, provide the driving force for proton movement back into the matrix (the protonmotive force) which will be utilized by a specific IMM protein (the mitochondrial ATP synthase) to convert ADP (adenosine diphosphate) to ATP (adenosine triphosphate). ATP thus produced is transported out of the mitochondria and is available to perform the work required by the cell.
The relative impermeability of the IMM to ion leak and the presence of energy conserving pumps and exchangers in the membrane allows for the efficient utilization of the protonmotive force for cellular energy production rather than expending it for reestablishing the IMM gradient. Despite this requirement for maintaining a high resistance membrane, the IMM contains a number of energy dissipating high conductance pathways for ion and/or solute movement. The physiological role of some of these pathways is apparent, for example, the pyruvate transporter is required to import substrate into the matrix for oxidative phosphorylation; however, in other cases, the physiological importance of IMM ion conducting pathways is still unknown. A well known example is the mitochondrial megachannel (MMC) or permeability transition pore (PTP). When activated, this large non-selective channel rapidly de-energizes the mitochondrion and has been implicated in several pathophysiological states. Other known high conductance pathways include the calcium uniport, the mitochondrial inner membrane anion channel, the mitochondrial uncoupling protein of brown fat mitochondria, and the mitochondrial ATP-sensitive potassium channel. A number of electrogenic (e.g., the adenine nucleotide transporter, the glutamate-aspartate transporter, the Na-Ca exchanger), proton-compensated electroneutral (e.g., the glutamate, pyruvate, and malate-citrate transporters) and electroneutral (e.g., the malate-phosphate, malate-ketoglutarate, carnitine, ornithine, and neutral amino acid transporters) are also present in the IMM and may influence the mitochondrial energy state. Although some of these channels and transporters have been well studied in isolated mitochondria, for lack of a useful index of mitochondrial activity in intact cells, much less is known about their regulation or sensitivity to pharmacological agents in intact cells.
The mitochondria are essential for efficiently providing ATP for carrying out the myriad functions of the cell, particularly in tissues with a high energy demand, such as muscle and brain. Consequently, defects in mitochondrial energy metabolism are usually associated with significant functional deficits or death. The pathophysiologies related to mitochondrial dysfunction can be either primary or secondary. In primary mitochondrial diseases, a genetic defect (either inborn or acquired) in a mitochondrial protein may lead to the incorrect assembly or catalytic activity of the protein, thus disrupting or impairing the entire biochemical pathway. Secondary mitochondrial disorders may arise from the accumulation of toxic products within the cell (including oxygen free radicals), the accumulation of inhibitory metabolites, or lack of cofactors required for mitochondrial metabolism.
Mitochondrial cytopathies of differing origin often lead to similar clinical symptoms. Commonly the disorders are first expressed in the most metabolically active tissues. They may present as muscle weakness and fatigue, mild muscle ache, or severe (and sometimes lethal) lactic acidosis during exercise. In many cases, these muscle deficiencies are also associated with central nervous system disorders, referred to as mitochondrial encephalomyopathies (e.g, KSS, Kearnes-Sayre syndrome; MERRF, myoclonus epilepsy with ragged red fibres; MELAS, mitochondrial encephalomyopathy/lactic acidosis/stroke). These disorders may arise from point mutations in or deletions of large segments of mitochondrial DNA. In many cases, the specific enzyme affected is known (e.g. pyruvate dehydrogenase deficiency). Similarly, defective nuclear encoded proteins involved in mitochondrial metabolism or drugs interfering with respiration (e.g., AZT or Adriamycin) can also lead to mitochondrial cytopathies.
Mitochondrial cytopathies can be classified by the site of the defect in mitochondrial oxidation. Defects in substrate transport (e.g. carnitine or carnitine-palmitoyl-transferase deficiencies), substrate metabolism (e.g. deficiencies in pyruvate dehydrogenase, pyruvate carboxylase, fatty acid oxidation, or organic acid metabolism), Krebs cycle activity (e.g. defects in oxoglutarate dehydrogenase or fumarase), the respiratory chain (NADH-Q reductase or cytochrome deficiencies), or energy coupling (ATP synthase defect, mitochondrial uncoupling diseases).
Cumulative alterations in mitochondrial metabolism have been suggested as an underlying cause of diseases associated with aging, including Alzheimer's and diabetes mellitus. Furthermore, mitochondria are the site of initiation of programmed cell death (apoptosis) and probably are the key factor in determining whether or not a cell will recover from an ischemic insult or proceed to necrosis.
Thus, mitochondria are central to the survival and function of the cell under normal conditions and play a major role in adapting to environmental stress.
Thus, it would be desirable to have a method of studying mitochondrial function as well as methods of assessing the effect of different chemicals on mitochondrial function. It would be particularly desirable to identify compounds that selectively modulate mitochondrial function. It would be also useful to detect compounds that affect mitochondrial K.sub.ATP channels.